1. Field of the Invention
This invention relates to peptide analogs that are interleukin-1xcex2 protease inhibitors. More particularly, the invention provides a-substituted methyl ketones derived from aspartic acid and the closed hemi-ketal forms thereof as inhibitors of interleukin 1-xcex2 protease.
2. Reported Developments
Enzymes involved in the catalytic degradation of proteins by hydrolyzing peptide bonds are known as proteases or proteinases. Proteinases are believed to be involved in various disease states including inflammation, metastasis, tissue damage, bone resorption and muscle degeneration in dystrophic diseases. Proteinases are divided into classes according to their catalytic mechanisms, such as serine-, cystein-, aspartic- and metallo-proteinases. For each class of proteinases, the catalytic site of the enzyme lies in the cleft on the surface of the enzymes in which reside the specificity subsites that bind amino acid side chains and the polypeptide backbone. In designing proteinase inhibitors, it is important to optimize the subsite binding characteristics with appropriate amino acid substrate analogs.
This invention relates to peptide substrates modified with affinity labels that inhibit interleukin-1xcex2 protease (hereinafter IL-1xcex2 protease). These inhibitors are thought to act by alkylating the cysteine sulfhydryl group (cys 285) within the catalytic site of IL-1xcex2 protease. Affinity labeling has been used since the 1960""s to prepare irreversible peptide-based inhibitors which act to alkylate the active sites of cysteine proteases. A variety of affinity labels and amino acid sequences have been synthesized to improve the binding of these modified peptide inhibitors to the enzyme""s active site. These affinity labels include peptidyl halomethyl ketones, peptidyl diazomethyl ketones, epoxysuccinyl peptides and peptidyl methylsulphonium salts as reviewed by D. Rich in Chapter 4 of xe2x80x9cProteinase Inhibitorsxe2x80x9d, Barret, A. J. and Salvesen, G., eds., Elsevier, 1986. More recently, peptide acyloxymethyl and aryloxymethyl ketons have also been described as affinity lables (Krantz, A. et al, Biochemisty, 30, p. 4678-4687, 1991). Current research (see for example European Patent Application, Pub. No. 015,748 A2; PCT International Publication No. WO 91/15577; Chapman, K. T., Biorganic and Medicinal Chem. Lett. 1992, 2, 613-618) has been directed towards understanding the enzyme binding specificity requirements in designing novel small molecular weight protease inhibitors that are efficacious, safe and have specificity for IL-1xcex2 protease which is believed to play an important role in many disease states (see Epstein, F. H., New Engl. Jrl. of Med., 32 p. 106-113, 1993).
Disease states in which IL-1xcex2 protease inhibitors may be useful as therapeutic agents include: infectious diseases, such as meningitis and salpingitis, septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, immune-based diseases, such as hypersensitivity, autoimmune diseases, such as multiple sclerosis; bone diseases; and certain tumors
The following publications illustrate that IL-1xcex2 inhibitors and antagonists are useful in modifying certain disease states in vivo.
1) IL-1 is present in affected tissues in ulcerative colitis in humans. In animal models of the disease, IL-1xcex2 levels correlate with severity. In the model, administration of 1-L-1ra reduced tissue necrosis and the number of inflammatory cells in the colon. Cominelli, F., Nast, C. C, Clark, B. D., Schindler, R., Llerena, R., Eysselein, V. E., Thompson, R. C., and Dinarello, C. A. xe2x80x9cInterleukin-1 gene expression, synthesis, and effect of specific IL-1 receptor blockade in rabbit immune complex colitisxe2x80x9d J. Clin. Investigations (1990) Vol. 86, pp, 972-980.
2) IL-1ra supresses joint swelling in the PG-APS model of arthritis in rats. Schwab, J. H., Anderle, S. K., Brown, R. R., Dalldorf, F. G. and Thompson, R. C. xe2x80x9cPro-and Anti-Inflammatory Roles of Interelukin-1 in Recurrence of Bacterial Cell Wall-Induced Arthritis in Ratsxe2x80x9d. Infect Immun. (1991) 59; 4436-4442.
3) IL-1ra shows efficacy in an small open-label human RA trial. Lebsack, M. E., Paul, C. C., Bloedow, C. C., Burch, F. X., Sack, M. A., Chase, W., and Catalano, M. A. xe2x80x9cSubcutaneous IL-1 Receptor Antagonist in Patients with Rheumatoid Arthritisxe2x80x9d Arth. Rheum. (1991) 34; 545.
4) IL-1 appears to be an autocrine growth factor for the proliferation of CML cells. Both IL-1ra and sIL-1R inhibit colony growth in cells removed from leukemia patients. Estrov, Z., Kurzrock, R., Wetzler, M., Kantarjian, H., Blake, M, Harris, D., Gutterman, J. U., and Talpaz, M. xe2x80x9cSupression of chronic myelogenous leukemia colony growth by interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptors: a novel application for inhibitors of IL-1 activityxe2x80x9d. Blood (1991) A; 1476-1484.
5) As in 4) above, but for acute myelogenous leukemia rather than chronic myelogenous leukemia. Estrov, Z., Kurzrock, R., Estey, E., Wetzler, M., Ferrajoli, A., Harris, D., Blake, M. Guttermann, J. U., and Talpaz, M. xe2x80x9cInhibition of acute myelogenous leukemia blast proliferation by interleukin-1 (IL-1) receptor antagonist and soluble IL1 receptorsxe2x80x9d. (1992) Bloods; 79; 1938-1945.
It is an object of the present invention to provide novel peptidyl substrate analogs modified with electronegative leaving groups that bind at the active site of IL-1xcex2 protease and inhibit IL-1xcex2 protease activity. IL-1xcex2 protease cleaves a biologically inactive 34 kD precursor of IL-1xcex2 to form the biologically active 17kD cytokine. This cleavage occurs at the peptidyl sequence of Val-His-Asp/-Ala-Pro-Val.
It is another object of the present invention to provide compositions comprising the above-referred to compounds.
It is a further object of the present invention to provide a method of use of the composition for the treatment of the above-identified disease states.
According to the present invention, there is provided a compound of the formula (I) and a pharmaceutically acceptable salt thereof: 
wherein
n=0-4; 
m=0,1;
R3=a singularly or multiply substituted aryl wherein aryl is a phenyl or naphthyl ring wherein the substituents are independently selected from the group consisting of
(1) H
(2) halogen
(3) OH
(4) CF3 
(5) NO2 
(6) OR5 
(7) COR9 
(8) NR6COR10 
(9) CONR5R6 
(10) SO2NR5R6 
(11) SO2R6 
(12) COOR11
xe2x80x83and
(14) lower alkyl and lower cycloalkyl;
R=(1) lower straight chain or branched alkyl, lower cycloalkyl
(2) (CR6R7)0-6-aryl
(3) (CR6R7)0-6-heteroaryl or
(4) (CR6R7)2-6xe2x80x94R8;
R6 and R7 are independently H, lower straight chain or branched alkyl, benzyl, aryl, cycloalkyl and aryl is defined as above and heteroaryl includes pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, isothiazolyl, benzofuranyl, isoxazolyl, triazinyl and tetrazolyl;
R8=(1) OCH2CH2OR6 
(2) OCH2CH2NR6R7 
(3) NR6CH2CO2R6
(6) NR6R7 wherein R6 and R7 are as above defined;
R9=(1) lower straight chain or branched alkyl, lower cycloalkyl
(2) (CR6R7)0-6-aryl;
(3) (CR6R7)0-6-heteroaryl; or
(4) (CR6R7)0-6xe2x80x94R8, wherein R6, R7 and R8 are as above defined;
R10=(1) R9 
(2) OR11 
(3) NR6R11,
wherein
R11=(1) lower straight chain or branched alkyl, lower cycloalkyl
(2) (CR6R7)1-6-aryl;
(3) (CR6R7)1-6-heteroaxyl; or
(4) (CR6R7)2-6xe2x80x94R8, and R6, R7 and R8 are as above defined;
R4=H or deuterium;
R2=(1) OR6 
(2) NR6OR7 or
(3) NR6R7, and R6 and R7 are as above-defined;
A=(1) an amino acid of the formula (II) 
xe2x80x83wherein R6 and R7 are as defined above;
R12 is independently
(1) H or
(2) (CR6R7)1-6xe2x80x94R13, and R6 and R7 are as above-defined;
R13=(1) H
(2) F
(3) CF3 
(4) OH
(5) OR11 
(6) NR6R14 
(7) cycloalkyl
(8) aryl
(9) heteroaryl
(10) SH
(11) SR11 
(12) CONR5R6 
(13) COOR5 or 
R14=(1) R7 
(2) COR10 
(3) SO2NR5R6 or

or
A=(2) an amino acid selected from the group consisting of 
R1 is an acyl group of the formula (III) 
wherein
R12 is
(1) OR5 
(2) NR5R6 
(3) R5 
(4) xe2x80x94CHxe2x95x90CHR5
xe2x80x83wherein R15=single bond, (CH2)2-6xe2x80x94NR6xe2x80x94, (CH2)2-6xe2x80x94Oxe2x80x94 and
R5 and R6 are as above defined; or
a sulfonyl group of the formula (IV) 
wherein 
xe2x80x83wherein R5 and R6 are as above-defined.
As used herein the term pharmaceutically acceptable salts include the acid and base addition salts.
The term acid addition salts refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid,citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
The term base addition salts include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaines, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylarine, choline and caffeine.
xe2x80x9cAlkylxe2x80x9d means a saturated or unsaturated aliphatic hydrocarbon which may be either straight- or branched-chain. Preferred groups have no more than about 12 carbon atoms and may be methyl, ethyl and structural isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl.
xe2x80x9cLower alkylxe2x80x9d means an alkyl group as above, having 1 to 7 carbon atoms. Suitable lower alkyl groups are methyl ethyl n-propyl isopropyl, butyl tert-butyl, n-pentyl neopentyl n-hexyl, and n-heptyl.
xe2x80x9cSubstituted phenylxe2x80x9d means a phenyl group in which one or more of the hydrogens has been replaced by the the same or different substituents including halo, lower alkyl nitro, amino, acylamino, hydroxyl, lower alkoxy, aryl, heteroaryl, lower alkoxy, alkylsulfonyl, trifluoromethyl, morpholinoethoxy, morpholino-sulfonyl, and carbobenzoxy-methyl sulfamoyl.
xe2x80x9cHalogenxe2x80x9d means chloride, fluoride, bromide or iodide.
xe2x80x9cLower cycloalkylxe2x80x9d means cycloalkyl having C3 to C6 carbon atoms.
The present invention also concerns the pharmaceutical composition and method of treatment of IL-1xcex2 mediated disease states or disorders in a mammal in need of such treatment comprising the administration of IL-1xcex2 inhibitors of formula (I) as the active agent. These disease states and disorders include: infectious diseases, such as meningitis and salpingitis; septic shock, respiratory diseases; inflammatory conditions, such as arthritis, cholangitis, colitis, encephalitis, endocerolitis, hepatitis, pancreatitis and reperfusion injury, immune-based diseass, such as hypersensitivity; auto-immune diseases, such as multiple sclerosis; bone diseases; and certain tumors.
In the practice of this invention an effective amount of a compound of the invention or a pharmaceutical composition thereof is administered to the subject in need of, or desiring, such treatment. These compounds or compositions may be administered by any of a variety of routes depending upon the specific end use, including orally, parenterally (including subcutaneous, intraarticular, intramuscular and intravenous administration), rectally, buccally (including sublinguaully), transdermally or intranasally. The most suitable route in any given case will depend upon the use, the particular active ingredient, and the subject involved. The compound or composition may also be administered by means of controlled-release, depot implant or injectable formulations as described more filly herein.
In general, for the uses as described in the instant invention, it is expedient to administer the active ingredient in amounts between about 0.1 and 100 mg/kg body weight, most preferably from about 0.1 to 30 mg/kg body weight for human therapy, the active ingredient will be administered preferably in the range of from about 0.1 to about 20-50 mg/kg/day. This administration may be accomplished by a single administration, by distribution over several applications or by slow release in order to achieve the most effective results. When administered as a single dose, administration will most preferably be in the range of from about 0.1 to 10 mg/kg of body weight.
The exact dose and regimen for administration of these compounds and compositions will necessarily be dependent upon the needs of the individual subject being treated, the type of treatment, and the degree of affliction or need. In general, parenteral administration requires lower dosage than other methods of administration which are more dependent upon absorption.
A further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a compound of the present invention in admixture with a pharmaceutically acceptable, non-toxic carrier. As mentioned above, such compositions may be prepared for use for parenteral (subcutaneous, intraarticular, intramuscular or intravenous) administration, particularly in the form of liquid solutions or suspensions; for oral or buccal administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops or aerosols.
When administered orally (or rectally) the compounds will usually be formulated into a unit dosage form such as a tablet , capsule, suppository or cachet. Such formulations typically include a solid, semi-solid or liquid carrier or diluent. Exemplary diluents and vehicles are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, mineral oil, cocoa butter, oil of theobroma, alginates, tragacanth, gelatin, syrup, methylcellulose, polyoxyethylene sorbitar monolaurate, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, and magnesium stearate.
The compositions may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington""s Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, Pa., 1985. Formulations for parenteral administration may contain as common excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like. Examples of vehicles for parenteral administration include water, aqueous vehicles such as saline, Ringer""s solution, dextrose solution, and Hank""s solution and nonaqueous vehicles such as fixed oils (such as corn, cottonseed, peanut, and sesame), ethyl oleate, and isopropyl myristate. Sterile saline is a preferred vehicle and the compounds are sufficiently water soluble to be made up as a solution for all foreseeable needs. The vehicle may contain minor amounts of additives such as substances that enhance solubility, isotonicity, and chemical stability, e.g., antioxidants, buffers, and preservatives. For oral administration, the formula can be enhanced by the addition of bile salts and also by the addition of acylcarnitines (Am. J. Physiol. 251:332 (1986)). Formulations for nasal administration may be solid and contain as excipients, for example, lactose or dextran, or may be aqueous or oily solutions for administration in the form of nasal drops or metered spray. For buccal administration typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinated starch, and the like.
When formulated for nasal administration the absorption across the nasal mucous membrane is enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, desoxycholic acid, chenodesoxycholic acid, dehydrocholic acid, glycodeoxy-cholic acid, and the like (See, B. H. Vickery, xe2x80x9cLHRH and its Analogs-Contraception and Therapeutic Applicationsxe2x80x9d, Pt. 2, B. H. Vickery and J. S. Nester, Eds., MTP Press, Lancaster, UK, 1987).